Kinetic Studies on Arachidonate 5-Lipoxygenase from Rat Basophilic Leukemia Cells

Abstract

Arachidonate 5-lipoxygenase of a 10,000 .times. g supernatant from RBL-1 cell homogenate was studied by a continuous assay measuring enzyme-catalysed oxygen consumption. Parallel HPLC and TLC analysis of arachidonic acid metabolites revealed that the oxygen consumption. measured is solely due to 5-lipoxy-genation of arachidonic acid. Oxygen consumption by this lipoxygenase was strictly dependent upon Ca2+, ATP and 5-HPETE. Removal of any of these three cofactors caused a complete inhibition of enzyme activity. Addition of the missing cofactor instantly restored the 5-lipoxygenase-dependent consumption of oxygen which remained linear for 10.sbd.20 s. Later on the velocity of the reaction decreased and after 2-3 min the enzyme became inactivated. Kinetic data were obtained from the initial velocity of the reaction using constant and saturating concentrations of CaCl2 and ATP. From Lineweaver-Burk plots substrate inhibition is evident for arachidonic acid concentrations greater than 45-50 .mu.M. Km(app) for arachidonic acid is 182 .+-. 16 .mu.M (mean .+-. SD, n = 5) and vmax(app) is 425 .+-. 140 nmol O2/(min .times. mg protein) (mean .+-. SD, n = 5).